N&V 110.pdf - NSPKU

NEWS AND VIEWS-ARTICLE...
the gene into hepatocytes or other tissues and to
restore phenylalanine metabolism. This research has
been greatly facilitated by the availability of the
PKU mouse for testing gene transfer methods. After
many years of work, recent experiments in the PKU
mouse have produced the most promising results
yet. But enthusiasm for these recent successes is
tempered by less than stellar results in the few gene
transfer clinical trials that have been attempted in
humans.
The primary site of phenylalanine metabolism is the
liver, so the liver is the natural target for gene
transfer. However, PAH enzyme is also produced
naturally in kidney and pancreas; transfer of the Pah
gene into these tissues or even tissues that don’t
normally contain PAH might lead to improved
phenylalanine metabolism and lower blood
phenylalanine levels.
In my laboratory, we have explored the possibility of
producing PAH enzyme in skeletal muscle or in
bone marrow in the PKU mouse, while the research
teams of Dr. Beat Thöny in Switzerland and Dr.
Thomas Jensen in Denmark have worked to produce
PAH in skin cells. All of these approaches have been
hampered by an inadequate supply of
tetrahydrobiopterin (BH4, the vitamin required for
PAH activity) in the target tissues. Work continues
to overcome this obstacle, but to date, these
approaches have not resulted in a permanent cure in
the PKU mouse.
Inserting The Gene With Viruses
The most promising results have come from gene
transfer experiments that target the liver. The liver
naturally produces its own BH4, so its supply will
not limit the effectiveness of gene therapy. Several
different methods for transferring genes into liver
cells exist, but the most successful attempts have
used Nature’s own method for injecting genes into
cells: infection with a virus. Viruses naturally infect
cells and carry in their own viral genes. After the
virus penetrates into a cell, the virus uses the
biochemical machinery of the host cell to make
multiple copies of itself. In the final step, the newly
formed virus particles break out of the host cell,
sometimes killing the host cell in the process, and
then continue on to infect other cells.
To use a virus as a vehicle for transferring
therapeutic genes into cells, many of the normal viral
genes are removed and replaced with the
therapeutic gene. These altered viral particles are
still capable of infecting cells, but because crucial
virus genes have been removed, they cannot
replicate themselves or produce new infectious virus
particles.
Adenovirus is a highly infectious virus that causes
the common cold. Adenovirus is capable of
infecting almost all tissues of the body and is quite
effective in penetrating liver cells. Adenovirus
vectors that have been altered to carry therapeutic
genes have been one of the most commonly
employed liver-directed gene transfer methods.
Researchers in Dr. Savio Woo’s lab designed an
adenovirus vector that carried the human Pah gene
and injected this virus into the liver of the PKU
mouse (Fang 1994). Liver PAH activity was restored
in treated mice by 4 to 7 days after injection of the
virus, but the amount of PAH activity varied
between 5-20% of normal. Blood phe levels were
completely corrected to normal in the animals that
had more than 10% PAH activity. Unfortunately,
blood phe began to slowly rise in all treated animals
and had returned to pretreatment levels by 2 weeks
after virus injection. Repeating the treatment had no
effect on blood phenylalanine levels.
The immune system in mammals has evolved to
quickly detect and destroy invading viruses. The
treated PKU mice had developed a very strong
immune response to the presence of the virus which
destroyed the infected liver cells and eliminated the
PAH enzyme. Following a second virus injection,
the immune system immediately recognized the
invading virus and eliminated it before it even had a
chance to infect the liver.
Injection into the liver of a similar adenovirus vector
has actually been used in a human clinical trial
designed to treat ornithine transcarbamylase (OTC)
deficiency, another inborn error of amino acid
metabolism. That trial was stopped because of a
similar problem with an immune reaction to the
virus. Jesse Gelsinger, an 18 year-old man with OTC
deficiency, received a large dose of an adenovirus
vector that had been engineered to carry the OTC
gene. Within hours of the infusion of the virus into
the liver, signs of liver damage began to appear. All
evidence pointed to a direct toxic effect of the virus.
Jesse eventually lapsed into a coma and died of
complete liver failure.
During the course of laboratory evaluation of
adenovirus, a new type of virus was discovered. This
previously unknown virus was found to be
contaminating many adenovirus cultures that had
been prepared from infected human tissues. This
new virus is now called adeno-associated virus
(AAV). Since the initial discovery of AAV, eight
different subtypes of AAV, all differing slightly from
each other and found in different mammals, have
been described. AAV can infect many different tissue
types but it cannot replicate itself unless an
adenovirus has also infected the same cell. AAV by
itself does not multiply within cells, kill the host cell,
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12 News & Views Issue 110